ALA-PDT 诱导 UVB 诱导的人皮肤成纤维细胞过早衰老中的氧化损伤和细胞凋亡。

ALA-PDT elicits oxidative damage and apoptosis in UVB-induced premature senescence of human skin fibroblasts.

机构信息

Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China.

出版信息

Photodiagnosis Photodyn Ther. 2016 Jun;14:47-56. doi: 10.1016/j.pdpdt.2016.02.005. Epub 2016 Feb 10.

DOI:10.1016/j.pdpdt.2016.02.005

PMID:26876682

Abstract

BACKGROUND

5-Aminolevulinic acid photodynamic therapy (ALA-PDT) has been used for the treatment of skin photoaging. It can significantly improve the appearance of fine lines, dotted pigmentation, and roughness of photoaged skin. However, the mechanisms by which ALA-PDT yields rejuvenating effects on photoaged skin have not been well elucidated. Thus, in this study we explored the effects of ALA-PDT in photoaged fibroblasts.

METHODS

We established a stress-induced premature senescence (SIPS) model by repeated exposures of human dermal fibroblasts (HDFs) to ultraviolet B (UVB) irradiation. Cells were irradiated by red light laser at 635nm wavelength (50mW/cm(2)). Intracellular protoporphyrin IX (PpIX) was detected by confocal microscopy. Intracellular reactive oxygen species (ROS) level and mitochondrial membrane potential (MMP) change were detected by fluorescence microscopy and flow cytometry. Morphological changes were observed by optical microscopy. Proliferative activity was measured by a cell counting kit-8 (CCK-8). Cell apoptosis was detected by fluorescence microscopy using Hoechst staining and flow cytometry using annexin V/propidium Iodide double staining.

RESULTS

Intracellular PpIX fluorescence in UVB-induced premature senescent HDFs (UVB-SIPS-HDFs) reached the highest intensity after incubation with 1.00mmol/L ALA for 6h (P<0.05). Compared with control group, intracellular ROS level, MMP, and apoptotic rate were increased (P<0.05) and proliferative activity was decreased (P<0.05) in UVB-SIPS-HDFs treated with ALA-PDT, which were positively correlated to ALA incubation time and red light laser dose.

CONCLUSION

Our study demonstrated that ALA-PDT elicits oxidative damage and apoptosis in photoaged fibroblasts in vitro, which may be the basis for the rejuvenating effects on photoaged skin.

摘要

背景

5-氨基酮戊酸光动力疗法（ALA-PDT）已用于治疗皮肤光老化。它可以显著改善细纹、点状色素沉着和光老化皮肤的粗糙度。然而，ALA-PDT 对光老化皮肤产生年轻化效果的机制尚未得到很好的阐明。因此，在这项研究中，我们探讨了 ALA-PDT 对光老化成纤维细胞的影响。

方法

我们通过重复暴露于人真皮成纤维细胞（HDF）于紫外线 B（UVB）照射来建立应激诱导的早衰（SIPS）模型。细胞用 635nm 波长的红光激光（50mW/cm²）照射。通过共聚焦显微镜检测细胞内原卟啉 IX（PpIX）。通过荧光显微镜和流式细胞术检测细胞内活性氧（ROS）水平和线粒体膜电位（MMP）变化。通过光学显微镜观察形态变化。通过细胞计数试剂盒-8（CCK-8）测量增殖活性。通过荧光显微镜用 Hoechst 染色检测细胞凋亡，用 Annexin V/碘化丙啶双染色通过流式细胞术检测细胞凋亡。

结果

用 1.00mmol/L ALA 孵育 6h 后，UVB 诱导的早衰 HDF（UVB-SIPS-HDF）细胞内 PpIX 荧光达到最强（P<0.05）。与对照组相比，ALA-PDT 处理的 UVB-SIPS-HDF 细胞内 ROS 水平、MMP 和凋亡率增加（P<0.05），增殖活性降低（P<0.05），与 ALA 孵育时间和红光激光剂量呈正相关。